The modification of enzymes with polyethylene glycol (PEG) is a technology that has markedly been developed in recent years to obtain adducts having valuable properties for the use both in the biomedical field and as novel biocatalysts, due to the presence of polyethylene glycol chains linked at the surface.
In fact, the enzyme-PEG adducts lose the major part of the typical properties of naturally occurring enzymes, such as immunogenicity and antigenicity, rapid clearance from circulation, easy degradability by proteases and instability in diluted solutions A. Abuchowski et al., J. B. C., 252 3582, 1977!, that often prevent their use in therapy.
In the use of enzymes in biocatalysis, the PEG-enzyme adduct acquires a quite different characteristic, i.e. the solubility in organic solvents, thus allowing a better use of the enzymes in converting liposoluble substrates Y. Inada et al., Thrends Biotech., 4 190, 1986!.
The properties of such novel biotechnologic products are due to the fact that PEG binds to the enzyme surface, thus protruding with its hydraration cloud toward the outer protein solvent, preventing the access of large molecules, such as proteolytic enzymes, as well as the recognition by the immune system. On the other hand, as PEG also has amphyphilic properties, the PEG-enzyme adduct can acquire solubility in organic solvents.
However, the polymeric cloud surrounding the PEG-enzyme adduct also limits the general use of said derivatives: in fact, the enzymatic activity is maintained toward small substrates, that can have access to the active site diffusing among the PEG polymer chains, but it is prevented toward large substrates, that cannot reach the active site due to steric hindrance.
There fore, convenient PEG-enzyme adducts are obtained with enzymes such as superoxide dismutase, catalase, asparaginase, arginase, urease, adenosine deaminase, phenylalanine ammonium liase etc., which are nowadays under pharmacological and clinical tests, but not with enzymes acting on large substrates such as proteins, nucleic acids and polysaccharides. In fact, substantial activity losses are de scribed following a PEG-modification of -trypsin, chymotrypsin, urokinase, ribonuclease, lysozyme and the like.
A proposed solution consists in preparing adducts having only a few polymer chains linked to the enzyme, thus decreasing the loss in enzymatic activity. However, this result, which can be attained carrying out the reaction in a PEG molar defect, suffers from drawbacks such as attainment of very heterogeneous products and poor reproducibility.